The principal long-term objective of this project is to provide a detailed biophysical and molecular understanding of exocytotic vesicle fusion and transmitter release in endocrine cells and nerve terminals. Upon electrical stimulation nerve terminals and endocrine cells release a variety of neurotransmitters and neuropeptides by an exocytotic mechanism. This process of neurosecretion is of outstanding importance in a broad range of physiological functions in the human body. It allows for synaptic transmission as well as release of hormones and neuromodulators and is thus an essential event mediating brain function, emotional response, behavior and various other physiological processes. A detailed understanding of the exocytotic event is of substantial interest, in particular for those diseases where neurosecretion is impaired. In exocytosis the vesicles are tethered at the plasma membrane and fuse with the plasma membrane allowing for release of the vesicle contents through the fusion pore. This project addresses 3 specific questions: 1) How is the mode of exocytosis (kiss-and run or full fusion) regulated? 2) What is the mechanism of fusion pore formation? 3) How are vesicles tethered to their target membrane before fusion is stimulated? We will investigate individual fusion events and individual tethering events using biophysical techniques. Fusion pore properties such as initial and mean conductance, conductance fluctuations, expansion rate, lifetime and permeability for transmitter molecules will be studied in chromaffin cells by patch capacitance measurements and analyzed statistically. Simultaneous measurements of fusion and release will be performed by "patch amperometry", a method that allows simultaneous recording of small capacitance changes and amperometric detection of released transmitter molecules. Biochemical manipulations and over-expression of wild type and mutant proteins implicated in fusion and release are used to investigate the role of specific proteins in determining the properties of the fusion pore. The biophysical properties of tethers are studied employing force measurements with optical tweezers using eosinophil granules as a model system.